Aim Toinvestigatetheeffectofrecombi-nant snake venom metalloproteinase inhibitor (rSVM-PI ) on neovascularization and its molecular mecha-nism.Methods Chickenchorioallantoicmembrane (CAM)assay was used to examine the antiangiogenic effect of rSVMPI.Alamar blue analysis was used to de-tect cell proliferation.Annexin V-FITC double labeling flow cytometry was used to assay cell apoptosis. Scratch marker was used to assay cell migration.Boy-den chamber analysis method was used to detect cells chemotaxis in vitro.Tube like structure(TLS)of HU-VECs was used to detect the ability of neovasculariza-tion in vitro.Real-time PCR and Western blot were used to assay the expressions of KDR and FGFR-1 inHUVECs. Results Thevasculardensityindex (VDI)of CAM was drastically decreased after rSVMPI treatment, chemotaxis of HUVECs in response of VEGF was inhibited in the presence of rSVMPI,TLS of HUVECs was less than control group.The expres-sions of KDR and FGFR-1 were down-regulated by re-al-timePCRandWesternblotassay.Conclusion rS-VMPI may inhibit neovascularization by blocking the VEGF-KDR or bFGF-FGFR signal transduction path-way.

Objective:To investigate the clinical significance of thrombospondin-2 (THBS2) mRNA expression in gastric carcino-ma and its relationships with clinicopathologic features, microvessel density (MVD), and matrix metalloproteinase-2 (MMP-2). Meth-ods:THBS2 mRNA expression was detected in 82 cases of gastric carcinomas and adjacent tissues using real-time quantitative fluores-cence polymerase chain reaction. The correlation of this expression with clinicopathologic features was also analyzed. Cluster of differ-entiation 34 (CD34) and MMP-2 protein expression was examined using an immunohistochemical Elivision method. MVD was deter-mined based on CD34-positive tubular structures. Results:The THBS2 mRNA expression level was significantly higher in the gastric carcinomas than in paraneoplastic tissues (P=0.002). The expression was associated with the depth of tumor invasion, MVD, and MMP-2 (P=0.02, r=0.35, P<0.01, and P=0.004, respectively) but not with patient gender, patient age, tumor size, histological type, and lymph node metastasis (P=0.53, P=0.53, P=0.21, P=0.84, and P=0.96, respectively). Conclusions: THBS2 may be significantly in-volved in the occurrence and progression of gastric carcinoma. The effects of THBS2 on gastric tumor growth and metastasis can be monitored by controlling the MMP-2 expression in the carcinoma. However, the specific functions and underlying mechanisms of TH-BS2 require further investigation.

Objective To investigate the effect of Alzheimer' s beta-amyloid (Aβ) on the production and the translocation in cytoplasm of retinoid receptor-α (RXRα). MethodsN2awt cells were treated with Aβ peptide or amyloid protein precursor(APP695) transfection. The nucleus were separated from the cytoplasm by kit. The quantity of RXRα in the nucleus and cytoplasm was detected by Westernblot. The translocation of RXRα in the nucleus and cytoplasm of above N2awt cells or of the cortex cells in the brains of Alzheimer' s disease (AD) patients and their normal control groups was observed by immune fluorescence. ResultsIn N2awt cells, the increasing APP or Aβ had no significant effect on the production of RXRα but resulted in RXRα exporting into cytoplasm, the ratio of RXRα in cytoplasm increased from 3.2％ (in control group) to 17.6％ (in APP+ group) and from 3.8％ (in control group) to 14.3％ (in Aβ + group) respectively; compared with normal cortex cells, the translocation of RXRα in the cytoplasm of the cortex cells in the brains of AD increased significantly. ConclusionAβ may affect RXRα exporting into cytoplasm.

Objective The aim of this study was to measure quantitatively the TSP-4 mRNA expression and its significance in gastric carcinoma was evaluated by correlating its expression with clinicopathological features, microvessel density(MVD) and MMP-9.Methods Eighty-two patients with gastric carcinoma were recruited in this study. TSP-4 mRNA expression in gastric carcinoma and adjacent tissue was detected by real-time PCR. CD34 and MMP-9 protein expression were examined by immunohistochemistry. MVD was determined according to CD34-positive tubular structures. Results The expression level of TSP-4mRNA in gastric carcinoma was obviously higher than those of adjacent tissue(P=0.03) and it was associated with tumor size, histological type, lymph node metastasis, MVD and MMP-9(P=0.002, P=0.031, P=0.014,r＝0.67 P<0.01, P=0.008),but not sex, age and depth of invasion. (P=0.53,P=0.57,P=0.15).Conclusion TSP-4 may play an important role in occurrence and progression of gastric carcinoma and that the effect of TSP-4 on tumor growth and metastasis may be exerted through regulation of angiogenesis and MMP-9 expression in gastric carcinoma.

Objective To screen the liver cell proteins interacting with the novel protein encoded by the 2.2 kb singly spliced variant of hepatitis B virus genome. Methods The splicing-specific gene TPss generated by the 2.2 kb singly spliced variant of HBV genome was amplified by PCR and cloned into the bait vector pGBKT7. After exclusion of self-activatian capacities of TPss protein, a two-hybrid library screening was performed using a pre-transformed human liver cDNA library to screen the liver cell proteins interacting with TPss. Mammalian two-hybrid assay was also done to further confirm the interactions between the bait and prey proteins in Huh7 and HepG2 hepatacytes. Results TPss gene with the size of 336 bp was successfully amplified and cloned, and the TPss protein expressed well in AHI09 yeast cells. Four liver cell proteins interacting with TPss, i. e. , cathepsin B, epoxide hydrolase 1, cathepsin D and fibrinogen gamma chain, were screened by yeast two-hybrid assay and further confirmed by mammalian two-hybrid assay. Conclusion TPss could interact with liver cell proteins.

Objective To activate microglia N9 cell through the oxygen deficit, and to discuss the influence to the N9 cell by ginsenoside Rb1, laying the foundation for the basic study and the clinical medicine development. Methods Through ginsenoside Rb1 intervention, the cell morphology the proliferation ability were observed, ELISA, fluorescent probe DAF-FM DA, Griess the reagent examination, were used to measure TNF-α, the O-2 output, the NO content change, chemiluminescence, the immunofluorescence method, and plastochondria membrane potential, were carried out to detect the cytochrome C content. Results Regardless of being preventive or medical gives, ginsenoside Rb1 can decline the NO,O-2,TNF-α high expression; and reduce the plastochondria membrane potential changing, the cytochrome C redistribution. Conclusion Ginsenoside Rb1 can decline N9 cell activation to a certain extent, reduce expression of the nerve toxic factor, and to stabilize mitochondrial membrane potential and distribution of cytochrome C.

OBJECTIVE To investigate the correlated resistant genes encoding ?-lactamases,aminoglycoside and genetic marker of integron and transposon in multi-resistant Pseudomonas aeruginosa(MRPA) isolated from clinical specimens and study their relationship by phylogenetic analysis.METHODS Twenty-one resistant genes,two integron-Ⅰ genes and one transposon genetic gene were analyzed by PCR and verified by DNA sequencing.Multi-resistant genes cluster analysis was performed by UPGMA.RESULTS The positive rates of CARB,oprD2,aac(3)-Ⅱ,aac(6′)-Ⅱ,ant(2″)-Ⅰ,intⅠ1,qacE△1-sul1 and merA in 20 strains of MRPA were 15%,100%,70%,15%,15%,85%,85% and 85%,respectively,and other genes were negative.It was classfied to three subgroup,by the multi-resistant genes cluster analysis.CONCLUSIONS MRPA isolated from clinical specimens has carried many resistant genes.The deficiency rate of oprD2 gene is very high.The positive rate of genetic mark genes about integron and transposon is very high.It may be the main multi-resistant mechanism of MRPA.Multi-resistant genes cluster analysis shows that there is clone transmission in MRPA and it can induce nosocomial infection prevalence.

Objective To analyze the effects of an inhibitor of the SH3 (Src homology) domains of Grb2 on the growth and proliferation of K562 cells. Methods The peptidimer [(VPPPVPPRRR)2-K], penetratin (RQIKIWFQNRRMKWKK) and peptidimer-c [poptidimer linked to penetratin: (VPPPVPPRRR)2-K-Aha-RQIKIWFQNRRMKWKK] were synthesized by solid-phase synthesis using Fmoc chemistry, and purified by high performance liquid chromatography (HPLC) on a C18 column. Purity was evaluated by HPLC, and the identity of the peptides was checked by electrospray mass spectroscopy (MS). A pull-down assay was used to observe the specific binding of peptidimer-c to the Grb2 of K562 cell lysates. The inhibition of peptidimer-c on K562 cell proliferation was evaluated by trypan blue exclusion assay, the cytostatic effect was tested by clonogenic assay, and the cytotoxicity was examined by WST-1 method. A further experiment was performed with clonogenic assay to analyze the co-effect of peptidimer-c respectively combined with Gleevec, Hydroxyurea and Cytarabine by Jing's method. Results The HPLC analysis showed only a simple peak, which means that the peptide is in high purity. MS analysis showed the peptides were coincided with the design. The molecular weight of peptidimer-c was of 4794.0 and that of the penetratin 2246.7. Pull-down assay demonstrated that the peptidimer-c, not the penetratin, could bind to Grb2 specifically. The trypan blue assay showed that the peptidimer-c could inhibit the proliferation of K562 significantly in a dose-dependent manner, even 3～6 h after the cells were exposed to the drug, and penetratin alone did not influence the cell proliferation. Gleevec inhibited the growth of K562 not only in a dose-dependent manner, but also in a time-dependent manner. WST-1 test showed the cytotoxieity of peptidimer-c or Gleevec on K562 cells, the IC50 of peptidimer-c was (17±2) μmol/L and the IC50 of Gleevec was (0.25±0.05) μmol/L. In the methylcellulose semi-solid medium system, the colony formation of K562 was greatly decreased by peptidimer-c as compared to the penetratin, and the colony number decreased as the dose of peptidimer-c increased. The IC50 value ofpeptidimer-c on K562 colony formation was (3.9±0.9) μmol/L, IC50 of Gleevec was (0.03±0.02) μmol/L, IC50 of Hydroxyurea was (15±7) μmol/L, and that of cytarabine was (0.014±0.012) μmol/L. There were synergistic effects of peptidimer-c with Gleevec, Hydroxyurea or Cytarabine on K562 by colonogenic assay. Combination of 1.5 μmol/L peptidimer-c and 0.05 μmol/L Gleevec showed synergistic effect on K562, as well as the combination of 1.5 μmol/L peptidimer-c and 0.006 μmol/L or 0.01 μmol/L Cytarabine. Conclusion These results suggested that peptidimer-c had an inhibitory effect on K562 cells and combination of peptidimer-c with other drugs would increase the anti-cancer effects.

Objective To investigate the anti-IFN-α effects of the novel protein TSR'r' encodedby the 458 nt-1308 nt spliced variant of hepatitis B virus genome,and to determine its functional domaias.Methods the TSR'r' gene(originated from open reading frame of HBV DNA polymerase,T represents terminal protein region,S represents the Spacer region,R'represents the truncated reverse transcriptase region,and r'represents the truncated RNaseH region)of the 458 nt-1308 nt spliced variant of HBV genome and its deletants were amplified by PCR and were cloned into the pcDNA3.1/HisC vector.The recombinant vector was transfected into Huh7 hepatocytes individually by FuGENE6 transfection reagent,and the expression of the fusion protein was detected by Western blot.Huh7 hepatocytes were co-transfected with p6 16CAT and the recombinant vector harboring either TSR'r'or the related deletant,and treated with IFN-α 2a 48 h post transfection.After 24 h stimulating.the cells were lysed and the intracellular CAT value was calculated.All data were processed with One-way analysis of variance(ANOVA).Resuits Recombinant vectors harboring either the TSR'r'gene or related deletant were constructed successfully,and the fusion proteins were expressed well in Huh7 cells.When Huh7 hepatocytes were co-transfected with p6-16CAT and TSR'r' recombinant.the intracellular CAT values reduced gradually as paralleled with the increasing amount of TSR'r'recombinant.Furthermore,as compared with the empty vector,intracellular CAT values also decreased significantly when the Huh7 cells co-transfected with recombinant harboring TP plus Spacer regions,while any of the other deletants(harboring either TP or Spacer region or neither)showed no significant difference.Conclusion The novel protein encoded by the 458 nt-1308 nt spliced variant of hepatitis B virns genome suppressed the response of Huh7 hepatocytes to IFN-α.and the N-terminal TP plus Spacer region was the functional domain of the protein for anti-IFN-α effects.

Objective To investigate the effects of the novel protein,TPds encoded by the 2.2 kb doubly spliced variant of hepatitis B virus(HBV)genome on the regulatory elements of HBV.Methods HBV promoters and enhancers were amplified by PCR and respectively cloned into the plasmid pGL3-basic to construct the recombinant firefly luciferase reporter vectors including pGL3-BCP(harboring basal core promoter),pGL3-CP1601(core promoter with enhancer Ⅱ),pGL3-XP(minimal X gene promoter),pGL3 XP1071(X gene promoter with enhancer Ⅰ),pGL3-SP1(promoter of large surface antigen)and pGL3-SP2 (promoter of middle surface antigen).The recombinant reporter plasmids were respectively co-transfected with either the pcDNA3.1/HisC-TPds(TPd8 expression plasmid)or pcDNA3.1/HisC(empty control)into Huh7 hepatocytes by FuGENE6 transfection reagent.Cells were lysed 48 h post transfection,the intracellulax luciferase activities were monitored and calculated by SPSS11.5 software.Results As compared with the empty control of pcDNA3.1/HisC.the intracellular luciferase activities were elevated when the Huh7 hepatocytes were co-transfected by pcDNA3.1/HisC-TPds with one of the recombinant reporter plasmids as pGL3-CP1601,pGL3-XP1071,pGL3-SP1,or pGL3-SP2,while no changes with pGL3-BCP or pGL3-XP.Conduslon TPds could transactivate the HBV regulatory elements a8 promoter SP1,SP2.and enhancer Ⅰ,Ⅱ,while no influences on basal core promoter and minimal X gene promoter.